The goal of this proposal is to close the gap in our understanding of how actin filament network remodeling, disassembly and turnover are regulated, and thereby clarify the mechanisms driving cell motility, cell morphogenesis, endocytosis, phagocytosis, and cytokinesis. A number of actin binding proteins besides ADF/cofilin have been genetically implicated in promoting actin turnover, but their specific functional roles, interactions, and mechanisms have remained elusive. This has left the molecular basis for dynamic remodeling and depolymerization of cellular actin arrays obscure. The research proposed here will first address how densely branched actin networks assembled by Arp2/3 complex such as those found at the leading edge of motile cells, sites of endocytosis, and trailing motile vesicles, organelles and pathogens are rapidly debranched and disassembled. These experiments will focus on the function and mechanism of a novel ADF/cofilin structural homologue we recently characterized, GMF, which binds to Arp2/3 complex and stimulates debranching (Gandhi et al., 2010), and how GMF activity is enhanced in the presence of coronin. This will include examining how GMF and coronin affect the conformation of Arp2/3 complex, and testing the hypothesis that ATP hydrolysis on Arp2 and Arp3 facilitates debranching by GMF and coronin. Further, we will explore the possibility that there is an analogy between the nucleotide state of Arp2/3 regulating coronin and GMF interactions in debranching and the nucleotide state of actin regulating ADF/cofilin and coronin interactions to promote severing. Second, we will address the basic mechanisms for stimulating actin filament severing and disassembly. These experiments will focus initially on our newly proposed roles for coronin in severing ADP-actin filaments and in spatially and temporally controlling ADF/cofilin severing effects (Gandhi et al., 2009). We will also address the contributions of two other conserved actin disassembly factors (Aip1 and twinfilin) in actin disassembly. Finally, we will use in vivo studies to determine how these five conserved disassembly factors (GMF, coronin, ADF/cofilin, Aip1, and twinfilin) act in concert to promote actin cytoskeleton remodeling and turnover in living cells. To achieve these goals, we will combine genetics, reconstituted in vitro motility assays, and novel multi- wavelength single molecule TIRF microscopy tailored to elucidate the mechanisms of multi-component regulatory systems. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo in motile cells to verify their biological importance and to assess their contributions to cell motility, in vivo actin turnover dynamics, and actin network ultrastructure (including branching) determined by cryo-electron tomography and correlative light and electron microscopy (CLEM). The Aims of the proposal are to: 1) Determine how the branched actin filament networks assembled by Arp2/3 complex are rapidly debranched by GMF and coronin. 2) Define the roles of coronin, Aip1 and twinfilin in actin filament disassembly. 3) Test the importance of debranching and turnover mechanisms for cell motility and in vivo actin network organization and dynamics.